3504

success rate of cervical cancer treatment is a big challenge in the medical field. Targeted therapy is the first choice in clinic8,9. The curative effect of current molecular targeting anti-apoptotic pro-teins, such as survivin and apollon on cervical cancer, is still not satisfactory. Therefore, it is urgently required to explore more effective mole-cular targets for the treatment of cervical cancer in clinic. Paclitaxel is a kind of important first-li-ne anticancer drug for the treatment of cervical cancer10. Paclitaxel catalyzes the rapid synthesis of excitron protein, which binds to the micro-tubules, leading to stabilization and prevention of microtubule depolymerization. Paclitaxel can inhibit mitosis, promote apoptosis, as well as inhibit tumor cell migration through directly or indirectly suppressing the expression and activity of myosin and actin11. After injected to the blo-od, paclitaxel is quickly decomposed into smaller albumin-paclitaxel polymer, and further binds to albumin GP60 receptor on the surface of vascu-lar endothelial cells12. Then, the albumin-paclita-xel is transported out of the blood vessel through endocytosis and aggregates around the tumors to regulate tumor formation. It was showed that the concentration of abraxane was 1.33-fold higher in the tumor tissue, while half in normal tissue com-pared with general solvent-based paclitaxel. Cur-rent guidelines recommend single use of abraxa-ne in the treatment of metastatic breast cancer13, pancreatic cancer14, and other solid tumors15. Up to now, however, the molecular mechanism of paclitaxel in inhibiting and eliminating cervical cancer cells remains poorly understood. Anti-a-poptosis protein survivin is a kind of protein kina-se involving in apoptosis, necroptosis, autophagy, and NF-κB signaling pathway16. It was speculated

Abstract. – OBJECTIVE: Paclitaxel is one of the common anticancer drugs in the treatment of cervical cancer, while the mechanism of re-straining and killing cancer cells is still unclear. This study aimed to investigate the molecular mechanism of paclitaxel in regulating prolifera-tion and apoptosis of cervical cancer Hela cells.

MATERIALS AND METHODS: Paclitaxel at 2 μmol/L was used to treat Hela cells for 48 h. MTT assay and flow cytometry were applied to test He-la cells proliferation and apoptosis respective-ly. Western blot was adopted to determine the ex-pression of survivin. SiRNA was performed to suppress survivin protein expression in Hela cells.

RESULTS: Paclitaxel restrained Hela cells growth and induced apoptosis. Also, paclitaxel treatment significantly reduced survivin protein expression in Hela cells. Moreover, survivin siR-NA transfection further promoted Hela cells apop-tosis after intervention by 2 μmol/L paclitaxel.

CONCLUSIONS: Down-regulation of survivin promoted paclitaxel-induced apoptosis of cervi-cal cancer Hela cells.Key Words:

Paclitaxel, Survivin, Cervical cancer, Hela, Apoptosis.

Introduction

Cervical cancer is a common reproductive system tumor in female with a higher morbidi-ty rate1,2. The pathogenesis of cervical cancer is very complicated and is thought to be influenced by viral infections, sexual behavior, and delivery times3-5. Also, genetic factors affect the develop-ment of cervical cancer6. Although the curative effect of combined therapy is significant, it has numerous shortcomings and side effects, such as bleeding7. At present, a variety of treatment strategies have been developed in clinical practi-ce7,8. However, how to improve the accuracy and

European Review for Medical and Pharmacological Sciences 2017; 21: 3504-3509

F. GU1, L. LI2, Q.-F. YUAN3, C. LI4, Z.-H. LI4

1Department of Obstetrics, Affiliated Hospital of Qingdao University, Qingdao, Shandong Province, China2Department of Pediatrics, Affiliated Hospital of Qingdao University, Qingdao, Shandong Province, China3Department of Gynecology and Obstetrics, Huangdao Qingdao District Wang Tai Center Hospital, Qingdao, China4Department of Gynecology, Qingdao Haici Hospital, Qingdao, Shandong Province, China

Corresponding Author: Lei Li, MD; e-mail: leiliqwe@sina.com

Down-regulation of survivin enhances paclitaxel-induced Hela cell apoptosis

Survivin mediates paclitaxel effect in Hela

3505

that survivin may play an important role in Hela cells apoptosis17. However, the exact mechanism by how survivin induces Hela cells apoptosis re-mains unclear. The aim of the present study was to investigate the specific regulatory mechanism of survivin on Hela cells apoptosis.

Materials and Methods

ReagentsCell apoptosis detection kit was purchased

from Beyotime (Shanghai, China). MTT, lipo-fectamine reagent escort™, and rabbit anti-hu-man survivin primary antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sur-vivin siRNA and control siRNA were designed and synthetized by Genepharma (Shanghai, China). The sequences were 5’GCGATGATTG-GTTATCGT3’, and 5’AAAATAGGTCATCA-TGGT3’, respectively. Dulbecco’s Modified Ea-gle Medium (DMEM) was provided by Beijing Dingguo Changsheng Biotechnology Co. Ltd (Beijing, China).

Cell CultureHela cells were purchased from ATCC. The cel-

ls were resuscitated and routinely cultured. Paclita-xel at 2 μmol/L or dimethyl sulfoxide (DMSO) was applied to treat the cells for 48 h.

Lipofectamine Escort™ TransfectionThe cells were seeded and transfected using

survivin siRNA and Lipofectamine reagent Escort™ according to the manual.

Flow CytometryCell membrane potential specific dye TMRE

and cell membrane phosphatidylserine specific dye FITC-Annexin V were applied to test the apoptosis of cervical cancer cell line by flow cytometry.

MTT AssayThe cells were seeded into 96-well plate at a

density of 10.000 cells/well for analysis of cell proliferation by methylthiazolyl tetrazolium bro-mide (MTT) assay.

Caspase-3 Activity DetectionThe cells were cracked on ice for 10 min and

added into the 96-well plate. Then, the chro-mophoric substrate was added to the plate for me-asuring the caspase-3 activity.

Western BlotThe cells were treated with lysis buffer and sepa-

rated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Primary antibodies for survivin and actin were applied for detection.

Statistical AnalysisAll data analysis was performed with SPSS18.0

software (SPSS Inc., Chicago, IL, USA). Data were represented as mean ± standard deviation (SD). Student’s t-test was performed for compa-ring the difference between two groups. One-way ANOVA and Dunnet post hoc test were perfor-med to assess the statistical significance among multiple treatment groups. A significant differen-ce was depicted as p < 0.05.

Results

Paclitaxel Inhibited Hela Cells Proliferation

The impacts of paclitaxel on Hela cell survival and proliferation were tested by MTT assay. As shown in Figure 1, paclitaxel significantly sup-pressed Hela cell growth (p < 0.05), suggesting it affects the proliferation of Hela cells.

Paclitaxel Reduced Cell Membrane Potential of Hela Cells

Mitochondrial membrane potential of Hela cel-ls was evaluated by flow cytometry after paclita-xel treatment. It was found that paclitaxel obviou-sly declined mitochondrial membrane potential of Hela cells (p < 0.05) (Figure 2), indicating that paclitaxel may induce the apoptosis of Hela cells.

Paclitaxel-induced Hela Cell ApoptosisTo confirm whether paclitaxel induces apop-

tosis of Hela cells, we measured the phosphati-

Figure 1. Paclitaxel inhibited Hela cells proliferation. * p < 0.05 vs. control.

F. Gu, L. Li, Q.-F. Yuan, C. Li, Z.-H. Li

3506

dylserine exposure of Hela cells after paclitaxel treatment. Results showed that after paclitaxel intervention, Hela cells exhibited markedly in-creased phosphatidylserine exposure (p < 0.05) (Figure 3), revealing that paclitaxel induces Hela cell apoptosis.

Paclitaxel Activated Caspase-3 in Hela Cells

Considering paclitaxel-induced apoptosis of Hela cells, caspase-3 activity was also measured. As shown in Figure 4, caspase-3 activity in Hela cells was obviously enhanced after paclitaxel tre-atment.

Paclitaxel Down-regulated Survivin Protein Expression in Hela Cells

Western blot was adopted to assess survivin protein expression in Hela cells. It was revealed that survivin protein level was down-regulated in Hela cells after treated with paclitaxel (Fi-gure 5).

Inhibition of Survivin Promoted Paclitaxel-induced Hela Cells Apoptosis

As shown in Figure 6, survivin level in Hela cells was suppressed after survivin siRNA tran-sfection, whereas, caspase-3 activity was enhan-ced in survivin siRNA transfected Hela cells treated with paclitaxel, revealing that down-re-gulation of survivin facilitated paclitaxel-induced Hela cells apoptosis.

Discussion

Paclitaxel is an important anti-cancer drug in clinic. In this study, we discussed the im-pacts of paclitaxel on the growth and apopto-sis of cervical cancer cell line Hela. Firstly, we explored the inf luence of paclitaxel on cervi-cal cancer Hela cells growth and survival and showed paclitaxel significantly decreased the proliferation of Hela cells, which is consistent with previous study. To evaluate the effect of

Figure 2. Paclitaxel reduced cell membrane potential in Hela cells. *p < 0.05 vs. control.

Survivin mediates paclitaxel effect in Hela

3507

paclitaxel on Hela cells apoptosis, f low cyto-metry and caspase-3 activity detection revealed that Hela cells exhibited mitochondrial mem-brane potential reduction and phosphatidylse-

rine exposure, suggesting that paclitaxel-in-duced Hela cells apoptosis possibly through activation of caspase-3, which was in accor-dance with previous studies17,18. However, va-

Figure 3. Paclitaxel-induced Hela cell phosphatidylserine exposure. * p < 0.05 vs. control.

Figure 4. Paclitaxel activated caspase-3 in Hela cells. *p < 0.05 vs. control.

Figure 5. Paclitaxel downregulated survivin protein expres-sion in Hela cells.

F. Gu, L. Li, Q.-F. Yuan, C. Li, Z.-H. Li

3508

rious apoptosis indexes and detection methods showed that under the similar concentrations of paclitaxel (2 μmol/L), Hela cells presented a higher sensitivity compared with other cancer cells. It may be caused by the fact that different cells have the diverse sensitivity to the same chemical drug. Apoptosis pathway mainly acti-vates caspase-8, while mitochondria-mediated intrinsic apoptosis pathway may activate ca-spase-3/719. In this study, paclitaxel activated caspase-3 of Hela cells, suggesting that the apoptosis of Hela cells induced by paclitaxel was possibly through mitochondria-media-ted intrinsic apoptosis pathway, which was in agreement of previous investigations20,21. As an inhibitor of apoptosis protein, survivin plays a critical role in apoptosis, necroptosis, autopha-gy and NF-κB signaling pathway16. Our results revealed that paclitaxel treatment significantly increased the apoptotic rate of Hela cells with down-regulation of survivin, indicating that knockdown of survivin contributes to cancer therapy. Based on our data, several investiga-tions could be carried out in the future. Cli-nical cervical cancer specimens in different stages could be collected to detect survivin expression to evaluate whether survivin is as-sociated with clinical stages. Different types of paclitaxel and similar drugs could be adopted to clarify the specific target and mechanism of paclitaxel on cervical cancer, thus providing more valuable information for clinical practi-ce. However, future studies are required to confirm our findings using survivin knockout mice.

Conclusions

Paclitaxel-treated Hela cells display reduced proliferation and increased apoptosis. Moreover, down-regulation of survivin facilitates paclita-xel-induced apoptosis of cervical cancer cell line Hela cells.

Conflict of interestThe authors declare no conflicts of interest.

References

1) Franco EL, SchLEcht nF, SaSLow D. The epide-miology of cervical cancer. Cancer J 2003; 9: 348-359.

2) Long hJ 3rD. Management of metastatic cervical cancer: review of the literature. J Clin Oncol 2007; 25: 2966-2974.

3) Ibeanu Oa. Molecular pathogenesis of cervical cancer. Cancer Biol Ther 2011; 11: 295-306.

4) Fang J, Zhang h, JIn S. Epigenetics and cervical cancer: from pathogenesis to therapy. Tumour Biol 2014; 35: 5083-5093.

5) ghIm SJ, baSu PS, JenSOn a. Cervical cancer: etiolo-gy, pathogenesis, treatment, and future vaccines. Asian Pac J Cancer Prev 2002; 3: 207-214.

6) mOOre ee, Wark JD, hOPPer JL, erbaS b, garLanD Sm. The roles of genetic and environmental factors on risk of cervical cancer: a review of classical twin studies. Twin Res Hum Genet 2012; 15: 79-86.

7) PeraLta-ZaragOZa O, bermuDeZ-mOraLeS Vh, Pe-reZ-PLaSencIa c, SaLaZar-LeOn J, gOmeZ-cerOn c, maDrID-marIna V. Targeted treatments for cervical cancer: a review. Onco Targets Ther 2012; 5: 315-328.

8) ZagOurI F, SergentanIS tn, chrySIkOS D, FILIPItS m, bartSch r. Molecularly targeted therapies in cer-vical cancer. A systematic review. Gynecol Oncol 2012; 126: 291-303.

9) PennatI m, FOLInI m, ZaFFarOnI n. Targeting survi-vin in cancer therapy. Expert Opin Ther Targets 2008; 12: 463-476.

10) hIrte h, kenneDy eb, eLIt L, Fung mFk. Systemic therapy for recurrent, persistent, or metastatic cervical cancer: a clinical practice guideline. Curr Oncol 2015; 22: 211-219.

11) tErziS aJ, thorSEn F, hEESE o, ViStED t, BJErkVig r, DahL o, arnoLD h, gunDErSEn g. Proliferation, migration and invasion of human glioma cells exposed to paclitaxel (Taxol) in vitro. Br J Cancer 1997; 75: 1744-1752.

12) tiruppathi c, Song w, BErgEnFELDt M, SaSS p, MaLik aB. Gp60 activation mediates albumin transcytosis in endothelial cells by tyrosine kina-se-dependent pathway. J Biol Chem 1997; 272: 25968-25975.

Figure 6. Paclitaxel-induced Hela cells apoptosis through activating survivin. *p < 0.05 vs. control. **p < 0.01 vs. control.

Survivin mediates paclitaxel effect in Hela

3509

13) ShigEMatSu h, kaDoya t, MaSuMoto n, SaSaDa t, EMi a, ohara M, kaJitani k, okaDa M. The effi-cacy and safety of preoperative chemotherapy with triweekly abraxane and cyclophosphami-de followed by 5-fluorouracil, epirubicin, and cyclophosphamide therapy for resectable bre-ast cancer: a multicenter clinical trial. Clin Bre-ast Cancer 2015; 15: 110-116.

14) SaiF Mw. u.S. Food and drug administration ap-proves paclitaxel protein-bound particles (Abraxa-ne®) in combination with gemcitabine as first-line treatment of patients with metastatic pancreatic cancer. JOP 2013; 14: 686-688.

15) grEEn Mr, ManikhaS gM, orLoV S, aFanaSyEV B, MakhSon aM, Bhar p, hawkinS MJ. Abraxane, a novel cremophor-free, albumin-bound particle form of paclitaxel for the treatment of advanced non-small-cell lung cancer. Ann Oncol 2006; 17: 1263-1268.

16) aLtiEri Dc. Survivin, versatile modulation of cell division and apoptosis in cancer. Oncogene 2003; 22: 8581-8589.

17) wang th, wang hS, Soong yk. Paclitaxel-indu-ced cell death: where the cell cycle and apoptosis come together. Cancer 2000; 88: 2619-2628.

18) Von haEFEn c, wiEDEr t, ESSMann F, SchuLzE-oSthoFF k, DorkEn B, DaniEL pt. Paclitaxel-induced apopto-sis in BJAB cells proceeds via a death receptor-in-dependent, caspases-3/-8-driven mitochondrial amplification loop. Oncogene 2003; 22: 2236-2247.

19) StraSSer a, O’COnnOr L, Dixit VM. Apoptosis si-gnaling. Annu Rev Biochem 2000; 69: 217-245.

20) kutuk o, LEtai a. Alteration of the mitochondrial apoptotic pathway is key to acquired paclitaxel re-sistance and can be reversed by ABT-737. Cancer Res 2008; 68: 7985-7994.

21) McconLEy DJ, DaViS Dw. Apoptosis and taxol the-rapy. Cancer Biol Ther 2002; 1: 118-120.